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089 THE ROLE OF 6-DEOXY-HEPTOSE SUGARS IN YERSINIA PSEUDOTUBERCULOSIS' PATHOGENESIS N Ho, D Ratnayake, C Creuzenet Yersinia pseudotuberculosis is one of the major causes of gastroenteritis, especially in third world countries. Treating disease caused by this ubiquitous foodborne pathogen would be an ideal goal in areas without sufficient sanitary care and health programs.
Department of Microbiology and Immunology, University of Western Ontario, London, Ontario
The lipopolysaccharide (LPS) is an essential virulence factor for Y. pseudotuberculosis and is implicated in its defense against the host immune system and invasion of intestinal epithelium. Its LPS contains unusual 6-deoxy-heptoses found also in several pathogenic bacteria, but not in mammals. The enzymes necessary for their synthesis could be targets for novel therapeutic agents such as inhibitors or vaccines. Little is known about the synthesis of these sugars, however we hypothesize that genes encoding a putative GDP-D-glycero-D-mannoheptose dehydratase (dmhA), and the associated reductase (dmhB) are involved in its biosynthesis, as they are encoded by an O-antigen biosynthetic cluster and are present in all strains that synthesize this unique sugar.
To test this hypothesis, we have created knockout mutants in each gene. Since 6-deoxy-heptose sugars branch off the main O-antigen chain in Y. pseudotuberculosis, we expect dmhA and dmhB mutants to still produce O-antigen but of a different composition than the wild-type. Indeed, we observe through silver staining and Western blot an alteration in O-antigen characteristics. GC-MS results show a loss of 6-deoxy-heptoses in the O-antigen of both mutants.
We are characterizing these mutants for their bile salt and novobiocin sensitivities, as well as for their invasion capabilities to Caco-2 cells. We are also attempting to complement the mutants to demonstrate gene specificity in our knockout phenotypes. We will cross-complement the mutants with equivalent genes from other foodborne pathogens that produce 6-deoxy-heptoses, most notably, Campylobacter jejuni. C. jejuni is the leading cause of world-wide enteritis, and its capsule polysaccharide is a major virulence factor that contains 6-deoxy-heptoses, therefore therapeutic agents based on Y. pseudotuberculosis DmhA and DmhB might also be efficient against C. jejuni.
The results indicate that dmhA and dmhB are involved in O-antigen synthesis, and may be essential for Y. pseudotuberculosis pathogenesis.